1. Field of the Invention
The invention relates to a method for processing of stable emulsions from whole-cell biotransformations.
2. Description of the Related Art
An aqueous-organic two-phase system is frequently used for biocatalytic conversion of a polar organic molecules [1-5]. This system allows the use and the accumulation of high concentrations of substrates and products having poor solubility in water. The organic phase, consisting of an a polar, non-toxic solvent or of a mixture of multiple solvents, serves as a substrate reservoir and/or as a product sink. Furthermore, the organic phase protects against toxic effects of substrates and products on the biocatalyst.
Furthermore, the characteristic distribution of substrates and products in the two phases can be utilized to prevent kinetic product inhibition, to steer equilibrium reactions into the desired direction, to increase enantioselectivity, and to monitor multi-step reactions.
Typically, such two-phase systems are strongly emulsified in order to achieve high mass transfer rates. The formation of stable emulsions is also promoted by high biocatalyst concentrations, especially if whole microbial cells are used. In this connection, high concentrations of macromolecular surfactant substances (cells, lipids, proteins, polysaccharides, biosurfactants, cell fragments) occur [6-9].
Because, in the case of two-phase bioprocesses, not only product isolation but also solvent recycling is essential for economic and ecological reasons, the two phases must be separated from one another after the biotransformation. This phase separation has proven to be difficult in the case of stable emulsions such as those that occur when using whole microbial cells. Various methods for phase separation, such as centrifugation, membrane filtration, filter coalescence, addition of demulsifiers or thermal methods yielded unsatisfactory results or were very complicated in terms of apparatus and time [7]. Complicated phase separation is considered a main limitation of industrial implementation of two-phase bioprocesses, with their great economic and ecological potential. In the sector of phase separation in the case of two-phase whole-cell biotransformations, there is therefore a need for innovation.
Typically, the systems from the biotransformation are at first roughly separated by means of centrifugation. Subsequently, multiple filtrations and (ultra)centrifugation steps are carried out, in order to achieve sufficient separation. The organic phase obtained in this very complicated manner is subsequently subjected to distillative or extractive processing, in order to separate out the valuable product. (In this connection, however, it is not possible to achieve adequate phase separation. Therefore it is not possible to separate the organic phase, which contains the valuable product, completely from the aqueous phase. This inability makes further processing significantly more difficult).
In the case of other separation methods, an attempt is made to purify the emulsion by means of distillation, after rough mechanical separation of other components, whereby problems occur due to fouling and two-phase states in the column. In the case of an enzymatic method, the emulsion is separated, with good results, by means of the use of hydrolases. Except for the method last mentioned, all previous methods are unable to achieve defined phase separation. Complete separation of not only the cells/cell components but also of the aqueous phase from the organic phase has not been possible until now.
Separation of the cell mass is of great importance in this connection, because this mass can lead to encrustations or blockages during subsequent process steps. Furthermore, no permanent separation of the phases can be achieved with the alternative solution approaches described. Aside from the great number of purification steps, another disadvantage of the previous methods is the use of a solvent for extraction, which might be required. Such solvent would have to be subsequently recovered.
The separation of aqueous-organic two-phase systems being discussed here will be described below, as an example, using the separation of coalescence-inhibited emulsions from two-phase whole-cell biotransformations, for example in a polar solvents. The reaction mixture present in this connection, after biotransformation has taken place, does not separate spontaneously and is present essentially as shown in FIG. 1, after it has been allowed to stand for a longer period of time. The mixture optically consists of three phases, whereby a milky, organic/aqueous emulsion forms the upper phase (I), which contains not only the organic solvent but also the educt, byproducts, and the product. Furthermore, this emulsion also contains dissolved components and surfactant substances (salts, nutrients, lipids, proteins, polysaccharides, biosurfactants, cells). The second optically identifiable phase (II) is an aqueous phase in which the nutrients required for cultivation (which are partly still present in the emulsion) are situated and from which the cells/biomass (III) settle in a third phase.
The complexity of the present reaction mixture becomes even clearer when one attempts to separate the two-phase system by means of conventional methods such as centrifugation. Thus, after extended centrifugation, the appearance shown in FIG. 2 is obtained. After extended centrifugation of the mixture from FIG. 1, the influence of the macromolecular surfactant substances present in the emulsion (lipids, proteins, polysaccharides, biosurfactants, cells, cell fragments) is clearly evident. Although the cells contained in the aqueous phase settle at the bottom of the vessel (IV), and the aqueous phase (III) has a sharp upper phase boundary surface, only insufficient separation into an organic phase (I) and an interphase or emulsion phase (II) can be observed in the emulsion (Phase I in FIG. 1).
A method for processing of a coalescence-inhibited emulsion having components from whole-cell biotransformations such as cells, soluble cell components, organic solvents and/or water is known from DE 10 2007 034 258 A1. In this method, the stable, coalescence-inhibited emulsion obtained after biotransformation is placed into a container with carbon dioxide in excess, and mixed for a predetermined period of time, at elevated pressure and elevated temperatures, whereupon the aqueous phase and the organic phase of the emulsion separate from one another, and the cells and cell components of both the aqueous phase and the organic phase precipitate in the region of their boundary surfaces or phase boundary surfaces, and are subsequently separated.
After the addition of carbon dioxide, preferably in excess (for example of about 3 mass parts of compressed carbon dioxide per mass part of emulsion) and preferably under a pressure of about 115 bar, for example, and at a temperature of about 45° C., for example, the emulsion is intensively mixed with the carbon dioxide, preferably for 2 minutes. The higher the temperature used here that is selected, the higher the pressure that should also be selected.
After the mixer is turned off, a sharp separation of the aqueous phase and the organic phase is subsequently observed. As a result, the cell components precipitate at the boundary surfaces of the phases (perhaps also at a boundary surface to a container or the like) both at the lower end of the aqueous phase and of the organic phase. These cell components can now simply be removed, because in contrast to the original emulsion, they sediment more rapidly. Even after the pressure is relaxed, the phases rapidly separate from one another even after they are mixed again repeatedly. The organic phase, which contains the valuable product, can subsequently be processed efficiently, for example by means of hypercritical extraction. A disadvantage of this method, however, is the relatively great effort in terms of equipment technology, because high-pressure equipment is required.